1. Field of the Invention
The present invention relates to a method for analyzing proteins in which an array of first capture molecules which are specific for peptide epitopes is employed, and to a corresponding capture molecule.
2. Description of the Related Art
Numerous methods of this type are known from the prior art, and they are used for the qualitative or quantitative analysis of proteins.
Examples of the areas of use of the known methods are protein analysis and protein detection. A further novel area of application of the known methods is the area of proteomics, a branch of research devoted entirely to proteins. One aspect of proteomics deals with comparing the protein composition in pathologically altered cells contrasted with normal, healthy cells. Investigations of this type are at present usually carried out by two-dimensional gel electrophoresis. Another field investigates the spatial structure of proteins, which is of interest in particular for drugs companies for developing novel medicaments.
In two-dimensional gel electrophoresis, the protein mixture to be analyzed is applied to a solid support, and a first separation takes place according to the content of acidic and basic amino acid units in the proteins via a pH gradient. The proteins are then fractionated in the second direction by an electric field, to result in a pattern of spots in which each spot represents one protein. It is possible by comparing the patterns of spots to analyze differences in protein compositions between healthy and pathologically altered cells. For identification, the protein spots are cut out and the proteins are fragmented by digestion with specific proteases. The masses of the fragments remaining after this treatment are characteristic of each protein.
In the method mentioned in the introduction for analyzing proteins, the detection takes place, for example, with protein-specific antibodies in array format.
To obtain such protein-specific antibodies, the applicant employs isolated, purified proteins, recombinantly produced proteins or chemically synthesized peptides derived from the protein sequence. The antibodies are obtained by immunization of laboratory animals with the proteins or peptides acting as antigens, and with adjuvants. It is also possible on the other hand to obtain the antibodies by in vitro methods as recombinant antibodies.
The peptides which are to be synthesized chemically are derived from known protein sequences, which are present, for example, in protein databases or can be found from nucleic acid databases, using software programs which allow theoretical predictions to be made about the protein structure and a possible antigenicity. Programs of this type are described, for example, by Lars Hennig: “WinPep-ein Programm zur Analyse von Aminosäuresequenzen”, BIOSPEKTRUM 4 (5), 1998, pages 49-50, or by Devereux et al.: “A Comprehensive Set of Sequence Analysis Programs for the VAX”, NUCLEIC ACIDS RESEARCH, volume 12, 1984, pages 387-395. With these programs it is possible to list all possible fragments, with molecular weight, sequence, sequence position and length, which are generated on cleavage of proteins for example by proteases or chemical agents. To predict the antigenicity, the probability with which the epitope is located on the surface of the protein is found on the basis of structural predictions; see, for example, Hopp and Woods: “A computer program for predicting protein antigenic determinants”, Mol. Immunol., volume 20, 1983, pages 483-489; further literature references are to be found in the worldwide web under the address www.expasy.ch.
Although the antibodies generated through application of these techniques are very good at recognizing the peptide employed for immunization, they often do not bind to the corresponding peptide epitope in the native protein. The reason for this may be, for example, that the peptide epitope in the native protein is not accessible to the antibody, for steric reasons, or that it is present, as a result of post-translational modification, in a modified form which cannot be derived from the database. A further reason may be that the peptide epitope is present in the native protein in a conformation which prevents antibody binding.
Thus, a disadvantage of this technique is the initial elaborate generation of a large number of peptide-specific antibodies against protein epitopes, which subsequently bind only poorly or not at all to the protein which is sought.
Proteins can be detected using such protein-specific antibodies, on the one hand, after fractionation by means of SDS-PAGE and transfer to membranes by Western blotting/immunoblotting techniques or by means of the ELISA technique. In the ELISA technique, the protein to be detected is bound, without previous separation of other proteins from a complete protein solution, directly to a highly specific antibody which is immobilized on a solid support. The protein solution is then washed off the solid support, and the protein which is bound, i.e. remaining on the support, is detected.
This detection takes place either with the aid of labeled second antibodies which are specific for the bound protein, or by competition of the analyte protein with labeled analyte protein which is added to the solution in defined amounts. This indirect detection makes use of the fact that unlabeled analyte protein from the sample and added labeled analyte protein compete in a manner which is defined by the law of mass action for the binding sites which are present. This results in the amount of bound, labeled analyte protein being inversely proportional to the amount of unlabeled analyte protein in the sample.
This method has on the one hand the previously mentioned disadvantage, namely that the preparation of the specific antibodies leads to a large number of antibodies which bind only poorly or not at all to the protein to be analyzed. A further disadvantage on use of the Western blotting technique is that the protein mixture fractionated by SDS-PAGE can in each case be tested only with one antibody or with differently labeled antibodies, respectively, in order to ensure distinguishability of the antibodies bound to the different proteins.
In the ELISA technique it is also always possible for only one antibody to be immobilized per analysis well and then incubated with the protein solution. This technique requires large amounts of sample because each analysis well must be incubated with one aliquot of the protein mixture. A further disadvantage in the known methods is that detection of the bound proteins requires specific second antibodies for each protein to be detected or large amounts of labeled analyte proteins for competition experiments.
As already mentioned in the introduction, the applicant also functionally immobilizes protein-specific antibodies differing in specificity in array format in rows and columns on a support material. The proteins to be analyzed are labeled and then incubated with the antibodies on the array. The proteins in the solution which are antigens for the immobilized antibodies then bind to the antibodies which are specific for them, resulting in spatially resolved specific protein binding.
It is possible on the basis of the known binding specificities of the immobilized antibodies and of the known positions of the respective antibodies in the array to determine the bound amount of the respective proteins in parallel. For this purpose, the bound amount of protein is measured via the labeling on the proteins by means of a spatially resolved detection.
Besides qualitative parallel detection of different proteins present in the sample solution, it is in addition to that possible by use of standard proteins to determine quantitatively the analyte proteins.
In contrast to the method described above, the advantage here is that a plurality of proteins can be detected in parallel in the same sample and in one well. However, the disadvantage is that the proteins to be analyzed must be labeled, for which purpose appropriate labels must be introduced on particular functional groups of individual amino acids of the proteins. The efficiency of such labeling reactions varies widely for different proteins, and it is determined by the particular immediate microenvironment of a functional group, i.e. by steric shielding, pH variation in the direct vicinity of the functional group due to neighboring groups, salts, solvents etc. In relation to the result, this means that the reactivity of chemically identical functional groups in a protein may vary widely, so that quantitative reaction of certain functional groups is very difficult.
In addition, the labeling reaction may lead to modification of amino acids within an epitope recognized by the specific antibody, which leads to loss of binding between protein epitope and antibody. However, this means that the protein is no longer detectable via the described assay method after the labeling.